Comparative ct method

Analysis of bacterial gene expression by qPCR has been described previously [21 (link)]. S. Typhimurium were grown in LB containing 0.3M NaCl [26 (link)] or low phosphate and magnesium-containing medium (LPM, pH 5.8) [28 (link)] for induction of T3SS-1 or T3SS-2, respectively. RNA extraction and qPCR were performed as described above. The data were analyzed using the comparative Ct method (Applied Biosystems). Transcription of the target gene was normalized to the levels of gyrA mRNA.

Total RNA from HeLa, HL60, U937, and THP-1 cell lines and from different subpopulations of PBMCs (pDC, NK, MO, CD40+, and B) was extracted using the TRIzol reagent (Invitrogen). For experiments with interferon stimulation, HL60 and U937 cells were either mock treated or incubated with 250 units/mL of IFN-α2a (Miltenyi Biotec) and harvested after 48 h. The RNA was converted to cDNA using an oligo(dT)18 primer and reverse transcriptase (Invitrogen). Reverse transcriptase quantitative PCR (RT-qPCR) reactions were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using SYBR Green I (Eurogentec). All reactions were performed in triplicate and experiments were repeated at least twice. Relative amount of mRNA was calculated using the comparative Ct method (Applied Biosystems): 2^−ΔΔCt = 2^{−[(Ct_target−Ct_housekeeping)  sample−(Ct_target−Ct_housekeeping)  control]}, where Ct is the threshold cycle and HPRT or β-microglobulins are the housekeeping genes.

Total RNA was extracted from the spleens of the vaccinated pigs using ISOGEN (Invitrogen). Reverse transcriptase-PCR (RT-PCR) was monitored using a one-step RNA PCR kit (Takara) with three pairs of primers according to the operating instructions. Primers are listed below: IFN-Υ (5′-GTTTTTCTGGCTCTTACTGC-3′; 5′-CTTCCGCTT TCTTAGGTTAG-3′) (Chaoprasid and Dersch, 2021 (link)); TNF-α (5-ACTGCACTTCGAGGTTATCGG-3′, 5′-GGCGACGGGCTTATCTGA-3′) (Meissonnier et al., 2008 (link)); interleukin (IL)-4 (5′-GTCTGCTTACTGGCATGTACCA-3′; 5′-GCTCCATGCACGAGTTCTTTCT-3′) (Duvigneau et al., 2005 (link)); GAPDH (5′-AACGACCCCTTCATTGAC-3′; 5′-TCCACGACATACTCAGCAC-3′).
Quantitative reverse transcriptase-PCR (qRT-PCR) was performed on the 7900HT Sequence Detection System (Applied Biosystems) using SYBR Green (Meissonnier et al., 2008 (link)). Each sample was analyzed in triplicate. Data were analyzed by a comparative CT method (Applied Biosystems). Transcript levels were calculated by normalizing to the levels of GAPDH mRNA. In addition, cytokines (IFN-γ, IL 4, TNF-α) in the spleen were analyzed in relation to the expression of antibodies (IgG, IgA) in the mucus, MLN and serum utilizing JMP software.¹

Total RNA was isolated from homogenized tissues (whole bodies or hemocytes) using the RNeasy micro plus kit (Qiagen) and quantified with a nanodrop (Agilent). First-strand cDNA was generated from 500 ng RNA using iScript cDNA synthesis kit, according to standard procedures (Biorad, California, USA). Gene specific primers were designed with Primer3 software [50 (link)]. Real-time quantitative PCR was performed on a DNA Engine 2 (MJ Research, Minnesota, USA) with qPCR MasterMix Plus for SYBR green I (Eurogentec, Belgium) using one internal reference transcript (elongation factor 1-α, NCBI RefSeq XM_001951252.2). Running parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 68°C for 30 s. All amplifications were performed in triplicate assays. Signal intensity was measured at the end of each elongation phase and results were analyzed using the Opticon 3.1 software provided by MJ Research. To assess the specificity of the PCR amplification, a melting curve analysis of the amplicon was performed at the end of each reaction and a single peak was always observed. Standard curves were established with four serial dilutions of first-strand cDNAs, ranging from 1/10 to 1/10000. ApMIFs expression levels were normalized to expression of the internal control elongation factor 1-α (EF1) using the comparative CT method (Applied biosystems, USA).